JPH06500468A - specific binding agent - Google Patents

Abstract

A reshaped human antibody or reshaped human antibody fragment having specificity for human polymorphic epithelial mucin (PEM) is produced by transferring the complementarity determining regions (CDRs) from a murine anti-HMFG hybridoma cell line HMFG1 into a human antibody variable region framework. The reshaped molecule can be used in the treatment or diagnosis of cancer.

Description

[Detailed description of the invention] 11. The stable host cell system according to claim 10, wherein the foreign gene comprises the following nucleic acid: Reotide sequence: i) GCCTACTGG ATA GAGii) GAG ATT TTA CCT GGA AGT AAT AAT TCT AGATACAAT GA G AAG TTCAAG GGC ■ TCCTACGACTTT GCCTG G TTT GCT TACiv) AAG TCCAGT CAG AGCC TT TTA TAT AGT AGCAAT CAA AAG ATCTAC TTG GCCv) TGG GCA TCCACT AGG GAA TCT vD CAG CAA TAT TAT AGA TAT CCT CGG A Host cell lines containing one or more CGs.

12. The stable host cell system according to claim 10, wherein the foreign gene is as shown in the accompanying drawings. A host cell line containing the entire nucleotide sequence shown in Figure 12.

13. The stable host cell system according to claim 10, wherein the foreign gene is as shown in the accompanying drawings. A host cell line containing the entire nucleotide sequence shown in FIG.

14. The stable host cell system according to claim 10, wherein the foreign gene is a) The following amino acid sequence: i) Ala Tyr Trp Ile Gluii) Glu Ile Le u　Pro　Gly　Ser　Asn　Asn　Ser　ArgTyr　Asn Glu Lys Phe Lys GlyiiD Ser Tyr Asp Phe Ala Trp Phe Ala Tyriv) Lys Ser S er　Gin　Ser　Leu　Leu　Tyr　Ser　5erAsn　Gi n Lys Ile Tyr Leu Alav) Trp Ala Ser Thr　Arg　Glu　5erVD　Gin　Gin　Tyr　Tyr　Ar g Tyr Pro Arg Thr; and b) The above encoded amino acid sequence has specificity for PE during its expression. A protein framework that can function as a CDR with A host cell system that also encodes.

15. The stable host cell system according to claim 10, wherein the foreign gene is as shown in the accompanying drawings. A host cell line encoding the entire amino acid sequence shown in FIG.

16. The stable host cell system according to claim 10, wherein the foreign gene is A host cell line encoding the entire amino acid sequence shown in FIG.

17. Plasmid psVgpt-HuVBHMFG1-HuIgGl.

18, Plasmid psVneo-HuVkBMFG1-HuCk019, synthesis Claim 17 or claims for production of specific binding agents, modified human antibodies or modified human antibody fragments Use of the plasmid according to claim 18.

20, Escherichia coli NCTC12411 strain.

21, Escherichia coli NCTC12412 strain.

22. against EIMFG, such as that contained in E. coli NCTC12411 strain. A DNA sequence encoding a modified human antibody heavy weight variable region with specificity.

23. Against EIMFG, such as that contained in E. coli NCTC12412 strain A DNA sequence encoding a modified human antibody light chain variable region having specificity.

24. Production using the expression vector contained in E. coli NCTC12411 strain A modified human antibody heavy weight variable region with specificity for HMFG that can be used.

25. Production using the expression vector contained in E. coli NCTC12412 strain A modified human antibody light chain variable region having specificity for flMFG, which can be used to

26, an improvement comprising at least one of the variable parts according to claim 24 or claim 25; Manufactured human antibodies or modified human antibody fragments.

27, the synthetic specific binding according to any one of claims 1 to 9 or claim 26; Combination drugs, modified human antibodies, or modified human antibody fragments can be used to slow or slow the growth of cancer cells. are bound or incorporated into drugs that can be stopped or detected in the human body. Synthetic specific binding agents, modified human antibodies or modified human antibody fragments conjugated to agents that can be released .

28. Synthetic specific binding agent, modified human antibody or modified human antibody fragment according to claim 27 in a pharmaceutically acceptable carrier.

29. Manufacture of therapeutic drugs to save humans from cancer or in-vivo diagnostic tests for humans. Any one of claims 1 to 9 or claim 26 for producing a dispensable composition. Use of synthetic specific binding agents, modified human antibodies or modified human antibody fragments as described above.

30. The synthetic specificity of claim 27 to a method of treating cancer or tomography in humans. use of synthetic binding agents, modified human antibodies or modified human antibody fragments.

Specification specific binding agent The present invention relates to specific binding agents, and more particularly to protein or non-binding agents. Polypeptide containing an amino acid sequence that specifically binds to proteins Regarding. More specifically, the present invention provides such specific binding agents by genetic engineering. Concerning how to produce.

Antibody structure Natural antibody molecules consist of two homologous heavy chain polypeptides and two homologous light chain polypeptides. These are covalently bonded by multiple disulfide bonds. . Figure 14 of the accompanying drawings illustrates the typical structure of antibodies of the IgG class. Each The peptide chains of are folded to form several independent domains. These 4 The N-terminal domain of the main peptide chain is rich in sequence diversity and is called the variable region (part 7). It will be done. The 7th part of one heavy chain (VH) and the 7th part of one light chain (VL) combine to bind antigen. form a joint. Consists of a combination of VH domain and vL domain The structural unit is called the Fv (variable fragment) of the antibody. The C-termini of the heavy chain and light chain are missing. Both have relatively conserved sequences and are called invariant regions.

The constant part of the heavyweights is composed of several domains, such as the γ-isotype ( The key constant region of IgG) consists of three domains (CHl, CH2, CH3) and CH It consists of a hinge region that connects the I domain and CH2 domain. two heavyweights The hinge part of is covalently bonded by a disulfide bond. The light chain has one constant domain The light chain constant domain is opposite to the CHI domain. Antibody portion The constant region of the offspring is mediated by complement-mediated cell lysis and antibody-dependent cell cytotoxicity (ADCC). It is involved in effector functions such as cleaning up foreign substances. With protease papain Digesting the antibody yields three fragments. One is CH2 domain and CH3 domain Fragments that contain FC fragments and are easily crystallized are called FC fragments. two remaining fragments are identical and are called Fab (antigen-binding) fragments, which contain VH and CHI domains. is combined with the entire light chain. Using pepsin, protein hydrolysis is Two Fab fragments remain connected via the hinge region, resulting in (Fab) Two fragments are obtained. Each domain has a distinct excitation at the gene level. Expressed in son.

Each variable part is a framework consisting of relatively conserved sequences. It contains three hyper-variable parts. These hypervariable regions interact with antigens It is a complementarity determining region (Complementarity Determining region). iningRegion (abbreviated as CDR). relatively preserved arrangement The column is Framework Region (abbreviated as FR) It is called. See the paper by Kabat et al. (1987). Antibody X-ray diffraction research Therefore, CDRs form a loop that protrudes from the tip of the antibody molecule, and FRs form a β-sea loop. It has been clarified that a framework structure consisting of a

modified antibody In certain embodiments, the present invention generally refers to [Altered J human antibodies] The complementarity-determining region of human immunoglobulin consists of a constant region and a framework region. (CDRs) that correspond to those found in non-human immunoglobulins. The present invention relates to immunoglobulins and partial fragments of such modified human antibodies.

General principles for producing such modified human antibodies and fragments 1 This policy is well known; References include Jones et al. (1986), Riecbmann et al. (1988) , Verboeyen et al. (1988) and European Patent Publication No. 239400. One example is linter.

Human proteins pose a risk of causing undesirable and adverse reactions when administered to patients. Modified human antibodies and their fragments can be used to treat human diseases in vivo ( It is particularly practical for diagnosis and treatment (in vivo), and the desired Specificity can be enhanced in host animals such as mice, from which a given Antibodies with specificity can be easily obtained. Variable region genes are from organisms other than humans. can be cloned from antibodies produced by humans (referred to as non-human antibodies), and Genetically engineer such CDRs into the mutant framework Modified human antibodies or fragments are thereby obtained. Get favorable results like this In order to It is necessary to sequence the entire variable population of non-human antibodies, preferably by determining the sequence of the entire variable population of non-human antibodies. Allows potentially important interactions between DR and framework to be identified.

human milk fat globule, HMFG and Antibodies against epithelial-derived neoplasms (abbreviated) (usually against defatted forms) neoplasm), particularly with breast, ovarian, uterine, and lung carcinomas . Taylor-Papadimitriou et al. (1981) and Arklie See the paper by et al. (1981). A well-characterized antibody (HMFGI) ) is known to bind to certain components of 1111FG, but These components have been found in certain body tissues, certain cancer tissues, and even urine; Polymorphic epithelial mucin; (abbreviated as PEM) (Gendler et al. (198g)). this It is believed that the peptide core of PEM is involved in the binding. Corresponding useful High specificity can be achieved by obtaining antibodies against cancer cells, e.g. breast cancer cell lines. Can be done.

European Patent Publication No. 0369816 (University of Melbourne, Xing et al.) describes human A monoclonal antibody specific for polymorphic epithelial mucin, which has a specific amino acid sequence. Antibodies that bind to are described. European Patent Publication No. 0369816 describes Ri “Humanizing” such antibodies by the method of Echmann et al. (1988) It has been suggested that it is possible. However, Xing et al. There is no equivalent description of the actual method for preparing antibodies.

Summary of the invention As one specific embodiment, the present invention has specificity for polymorphic epithelial mucin. specific binding synthetic polypeptides, in particular the CDs shown in Figures 1 and 2 of the accompanying drawings. A specific product containing one or more R and having anti-human milk fat globule (HMFG) specificity synthetic binding polypeptides are provided. The term "synthetic" refers to such polypeptides. Tido has been produced using recombinant DNA technology and, to the extent that it exists, is not naturally occurring. Minimally different from a specific binding agent of the same specificity that exists or is naturally derived It means that. Synthetic polypeptides also have amino acid sequences that are artificially assembled. It can also be produced by creating new or naturally occurring molecules.

Synthetic polypeptides may be equivalents of normal intact antibodies. may be equivalent to a multi-chain or single-chain fragment of such an antibody, or A substance containing one or more sequences that merely confers the desired specific binding ability. Good too.

As one important embodiment of the present invention, a modified human antibody with anti-PEM specificity is provided. body or modified human antibody fragments, in particular one or more of the CDRs shown in Figures 1 and 2 of the accompanying drawings. modified human antibodies or modified human antibody fragments containing more than that and having anti-HMFG specificity. Serve pieces. Preferably, such engineered antibodies or fragments of the invention are human heavy variable antibodies. 1 of the accompanying drawings. this Alternatively or concurrently, the modified antibodies or fragments of the invention may contain human light chain variable region fragments. It includes all three CDRs shown in Figure 2 of the accompanying drawings in its framework.

Another embodiment of the present invention is the protein shown in FIG. 12 and/or FIG. 13 of the accompanying drawings. It is a modified antibody or modified antibody fragment that contains the original sequence.

Other important embodiments of the invention are shown in FIGS. 12 and/or 13 of the accompanying drawings. Expression vectors incorporating the DNA sequences shown in Figures 1 and/or 2 of the accompanying drawings. DNA sequences encoding one or more protein sequences designated as CDRs This is an expression vector that incorporates

One important aspect of the invention is to induce production of specific binding agents of the invention by host cells. A stable host cell line containing a foreign gene that causes Such cell lines are Some of the amino acid sequences shown as CDRs in Figure 1 and/or Figure 2 of the accompanying drawings. both encode one sequence, and the encoded amino acid sequence in parentheses is expressed during its expression. Protein fragments that can function as CDRs with specificity for HMFG A stable host cell line containing a foreign gene that also encodes the framework can be done.

The invention further provides immortalized antibodies capable of producing modified antibodies or fragments of the invention. mammalian cell lines or yeast or other eukaryotic cells or prokaryotic cells such as bacteria. provide cells.

Another important aspect of the invention is D1. Anti-Bll [FG monoclonal antibody] A synthetic specific binding agent with specificity comparable to that of rBMFGIJ, a modified human anti- body or modified human antibody fragment.

The present invention further provides two new plasmids pSVgpt-BuVHHMFGl. - Provides HuIgGl and psVneo-11uVkHMFG1-HuCk However, these plasmids contain synthetic specific binding agents, modified human antibodies, or modified human antibody fragments. Can be used to produce pieces.

Each of these plasmids is a novel Escherichia coli strain oli) strains NCTC12411 and NCTC12412.

Other aspects of the invention are as follows.

a) Specificity for HMFG, such as that contained in E. coli NCTC12411 strain A DNA sequence encoding a modified human antibody heavy weight variable region having a specific sex.

b) Specificity for HMFG, such as that contained in E. coli NCTC12412 strain 1. A DNA sequence encoding a modified human antibody light chain variable region having a specific property.

C) Production using an expression vector contained in E. coli NCTC12411 strain A modified human antibody heavy weight variable region having specificity for HMFG.

d) Production using an expression vector contained in E. coli NCTC12412 strain 1 [Modified human antibody light chain variable region with specificity for FG.

e) a modified human antibody comprising at least one of the variable regions of C) or d) above, or Modified human antibody fragment.

Certain embodiments of the present invention are therefore shown in FIGS. 1 and 2 of the accompanying drawings. II, CDR group with amino acid sequences identified as CDR2 and CDR3 (this They can be cloned from, for example, mouse anti-1111FG immunoglobulin. ) modified human antibody or modified human having anti-BMFG specificity incorporating a certain combination of It is an antibody fragment. Please note that Figures 1 and 2 of the attached drawings were created by us this time. A heavyweight of mouse anti-HMFG monoclonal antibodies cloned and sequenced The variable region (VH) and light chain variable region (Vk) are shown. If the antibody is intact, or is a fragment comprising at least one heavy chain variable region and at least one light chain variable region; If the modified antibody or fragment is derived from a source other than human (referred to as non-human origin) Preferably, it contains all six CDRs. For maximum connectivity, use this Preferably, the relative positions of the CDHs of these CDHs are as per the arrangement found in the original non-human antibody. For example, each CDR of vH should be within the human VH framework) , in the natural order found in non-human antibodies.

As will be clear to those skilled in the art, these CDR sequences and their surrounding Framework sequences can be used in a variety of ways without appreciably reducing the basic specific binding strength. Various modifications and mutations can be made. Such various modifications and mutations are caused by genetic It may exist at the child level or in the amino acid sequence; may exist in Therefore, the present invention provides the ability to Synthetic (modified) antibodies and fragments functionally equivalent to antibodies or antibody fragments with amino acid sequences. Also includes pieces.

The present invention is also applicable to the production of bispecific antibodies having two Fab parts with different specificities. Can be used. In this case, one of the peculiarities is that the CD shown in Figure 1 or Figure 2 of the accompanying drawings provided by a modified human variable region into which at least one of H.

The present invention relates to so-called single chain antibodies (for example, Genex European Patent Publication No. 28160 It can also be applied to the production of polysaccharide-binding antibodies (such as those disclosed in No. 4). ybritech (see European Patent Publication No. 315456) and other modified antibodies. It can also be applied to production.

Any human constant region (e.g. γ1.12, γ3 or 14 type) can be used. It's okay.

Antibodies that retain useful specific binding properties include (Fab) 2, Fab, Examples include Fv, VH or Vk fragments.

These can be obtained from intact modified antibodies, for example by protease digestion. It can also be produced as is through genetic engineering.

Practical applications of the invention One of the important aspects of the present invention is to make the modified human anti-HMFG antibody or fragment described above, It is bound to drugs that can slow or stop the growth of cancer cells or contains such drugs, or contains a contrast agent that can be detected in the human body. It is. The invention further provides that any such combination is pharmaceutically acceptable. in a carrier such as saline, plasma extender or liposomes. Also included are injectable compositions comprising.

The present invention further provides the use of the above-mentioned modified human anti-HIl [FG antibody or fragment for human cancer. It also includes use in the treatment of or in methods of imaging. present invention furthermore, the above-mentioned antibodies or Use of the fragments as well as the above antibodies in the manufacture of diagnostic compositions for human in vivo diagnosis. or the use of fragments.

The Fc region of an antibody itself has functions such as complement lysis as well as antibody-dependent cellular cytotoxicity. It can be used to inhibit the growth of cancer cells through mechanisms available to the body.

In this embodiment, there is no need to attach any further agents to the modified antibody. .

Specific examples of drugs that can inhibit the proliferation of cancer cells include 90Y and +31 Any radioactive isotope, drug such as methotrexate, poison such as ricin, or any of them. moiety as well as, for example, at the antibody binding site, to convert an inactive drug into an active drug. An example is eel enzyme.

Specific examples of contrast agents include radioactive materials that generate T-rays such as 1ljIn and 99TC. isotopes, radioactive isotopes that generate positrons such as 64Cu, and as contrast agents for X-rays. Immobile substances such as barium for action and gadolinium for MNR/ESR scanning Examples include state reagents.

In order to bind a metal reagent such as a radioactive isotope to the specific binding agent of the present invention, It may be necessary to use coupling or chelating agents. many suitable keys Rate agents have been developed, e.g. referenced in US Pat. No. 4,824,986. , No. 4831175, No. 4923985, No. 4622420, etc. can be mentioned. Techniques relating to the use of chelating agents are described, for example, in U.S. Pat. No. 454106 and No. 4722892, and 11oi et al. (1988), M cCall et al. (1990), Deshpande et al. (1990) and Meare It is described in the paper by S et al. (1990).

Use of radiolabeled antibodies and fragments for human cancer imaging and treatment This is described, for example, in European Patent No. 35265. radioactive like this Labeled cancer-specific antibodies or fragments can be used in so-called subtractive imaging ( A control pack ground for subtraction imaging) Effective when used with non-specific reagents radiolabeled with another isotope to give be.

The antibody reagent of the present invention can be used to detect PEII production, for example, by serum tests or tomography. It can be used for the identification of PEl-producing cancers and/or for the treatment of PEl-producing cancers. like this These cancers are found, for example, in breast, ovarian, uterine, and lung carcinomas, and may cause pleural effusions. Sometimes it appears as a liquid.

Production of modified antibodies The V■ and VL domains are conventionally called CDIII (Kabat (1987)). is the only feature that needs to be imported from the non-human monoclonal antibody. Not necessarily the target part. In some cases, human antibodies modified with non-human framework sequences What is the specificity and/or affinity of the modified human antibody when stored in the body? It may also improve the performance of the antibody.

The aim is to target CDRs retained by their connections with framework residues. The goal is to preserve the associated important three-dimensional protein structure.

A typical starting point for producing engineered antibodies of the invention is 111 [FG or PE11 a non-human host animal (e.g. mouse) that expresses antibodies with specificity for (preferably an immortalized cell line). Such cell lines can be used, for example, in Even with hybridoma cell lines prepared using conventional monoclonal antibody technology, good. The expressed antibody has high affinity and specificity for HMFG. preferable. affinity and/or Otherwise, it is expected that some loss of specificity may occur. Highly specific anti- By selecting the body as the parent antibody, the final modified antibody or fragment It is also more likely to exhibit effective binding properties.

The next step is to clone cDNA from cells expressing the selected non-human antibody. , at the stage of sequencing and identifying variable region genes including CDR-encoding sequences. be. The experimental techniques used here remain laborious, but current technology It can be considered as an established routine.

Modified human antibodies (complete) or both heavy and light chain variable domains If the purpose is to produce fragments of such antibodies that contain both It will be necessary to sequence the cDNAs associated with these domains.

Once the relevant cDNA sequences have been analyzed, at least the variable domain of the antibody can be analyzed. It is required to prepare a replicable expression vector containing the encoding DNA. . Here, this variable domain combines human framework regions with a given non-human anti-HM together with one or more CDRs derived from the FG antibody. each vector The DNA sequence in the gene contains the appropriate Regulatory sequences, especially leader sequences and promoters operably linked to variable domain sequences. should include the following. Exemplary Procedures for Producing Modified Antibodies or Fragments of the Invention , two types of such expression vectors (i.e., one containing the modified human light chain DNA sequence) It is necessary to prepare two types: one that contains the DNA sequence of a modified human heavyweight and one that contains the DNA sequence of a modified human heavyweight. Sometimes it's necessary. These expression vectors can be transformed into a given cell line to generate modified antibodies. It must be such that it causes the production of bodies or fragments.

Such a cell line can be, for example, a stable non-producing myeloma cell line. It takes Specific examples of myeloma cell lines (such as N20 and 5p2-0) are readily available from commercial vendors. . Another option is to use bacterial systems such as E. coli as expression vehicles for engineered antibodies or fragments. It is to use it. The final step in this procedure is therefore to use one or more expression vectors. transform a given cell line or organism using a vector, and then transform the transformed cells. culturing cell lines or organisms to obtain modified human antibodies or fragments.

For illustrative purposes only, suitable expression vectors will be prepared hereinafter. We will introduce the details of the steps that can be taken. In a laboratory with appropriate equipment The handling of DNA material is already a well-developed technology and is needed. The operating procedures involved are within the scope of those skilled in the art. Perform these operations Genomic libraries, cDNA libraries, plasmids, and restriction enzymes necessary for A large number of suitable specimens of base materials, various reagents and culture media are available from research material sales companies. It is commercially available from.

For example, for genomic and cDNA libraries, C1ont It can be purchased from ech Laboratories Inc. onwards The steps illustrated in are provided merely as a guide for the reader of this specification. Thus, the invention is in no way dependent on particular starting materials. In fact, those skilled in the art A wide variety of materials can be selected and can be easily obtained in a scientific environment. Known techniques can be adopted and utilized based on the experience and materials available. for example, A large number of plasmids fall into this category, but these are widely used in the relevant scientific community and These materials can now be considered commonplace.

Example The methods used to prepare the modified anti-HMFG human antibodies are described below for illustrative purposes only. will be described in detail with reference to the accompanying drawings.

Here, the attached drawings will be explained.

Figure 1 shows cDN^ sequences encoding mouse heavy variable regions with anti-HMFG specificity. This shows the columns. Three classical CDI+ locations matched with cDNA code Shown together with the amino acid sequence.

Figure 2 shows the cDN encoding the mouse light chain variable region with anti-HIIFG specificity. This shows the arrangement.

Figure 3a shows a synthetic modified VH gene with HMFGI specificity containing three fragments. This shows the basic structure of the gene (HuVHIcooBMFG1 gene cassette). be.

Figures 3b to 3d show the sequence of each fragment in Figure 3a for each fragment. This is shown together with the oligonucleotides used in the construction.

Figures 4a, 4b, and 40 collectively show the cDNA introduced modified human heavyweights of Figure 1. 2 shows a route by which expression vectors encoding the expression vectors can be prepared.

Figures 58 and 5b show generally similar cDNA-introduced modified human light chains of Figure 2. This shows a transformation route by which expression vectors encoding Ru.

Figure 6 shows a plus sequence containing the enhancer sequences used in the routes of Figures 4a-5b. Mid pUc12-IgEnh is shown.

Figure 7 shows the source of plasmid pBGS18-HuIgG1 used in the route of Figure 40. This is what is shown.

Figure 8 shows the source of plasmid pBGS18-rouCk used in the route of Figure 5b. It is something that

Figure 9 shows the two types of synthetic oligomers used to clone the cDNA sequences in Figures 1 and 2. The oligonucleotide sequence engineering and II are shown.

FIG. 10 shows that M13mp9HuVHLYS, respectively, in the route shown in FIG. Two types of synthetic oligos were used to introduce the KpnI and 5alI restriction enzyme sites. nucleotide sequences III and IV.

Figure 11 shows that on the human VK REI framework region in the pathway shown in Figure 5a. Three types of synthetic oligonucleotides used to import Vk HMFGI CDRs into Reotide sequences VI, VII and VIII are shown.

Figures 12 and 13 show the obtained modified human heavyweight variable region and light chain variable region, respectively. The cDNA and amino acid sequences of the region are shown.

Figure 14 shows a schematic diagram of the structure of a typical antibody (immunoglobulin) molecule. It is.

Figure 15 graphs the relative specific anti-BMFGI binding activity of the obtained modified human antibodies. This is shown in a flat format.

The experimental procedures required to carry out the present invention are not in themselves unusual techniques. Black For example, Verhoeyen et al. (1988), Ri Echmann et al. (1988) and European Patent Publication No. 239400 (W Inter). Modified human heavyweight variable region genetics The child's "Cde novo" J synthesis (see Figures 3a to 3d) is different from the conventional method. A set of long overlapping oligonucleotides (see Jones et al. (1988)) (Reference) was used. Laboratory equipment and reagents for long-chain oligonucleotide synthesis are Chain lengths of sequences that are readily available and that can be synthesized as technology in this field develops. is gradually getting longer.

7 detailed experiments covering all basic aspects of recombinant DNA technology, examples For example, Sambrook et al.'s llocular Cloning J (1 989) etc. are also available.

According to the present invention, the antigen-binding region of a mouse anti-HMFG antibody (HMFGI) can be converted into a human antibody. moved into the teamwork area. The obtained modified human antibody (named HuHi [FGl) ) has binding properties similar to those of the original mouse antibody.

Such modified antibodies can be used for in vivo diagnosis of human cancers (e.g., ovarian cancer, breast cancer, etc.). It can be used for treatment of It is believed that this can at least reduce the problem of immune reactions that occur. Hale (1988) report that a modified CAMPATH-1 antibody offers similar advantages. It has been revealed that

anti-HMFG antibody rHMFGIJ (Taylor-Papadimitr iou et al. (1981) and Arklie et al. (1981)). mRNA was isolated from a mouse hybridoma cell line. CHI exon respectively and oligonucleotides I and II complementary to the 5′ end of the Ck exon (Figure 9 The first stage cDNA was synthesized using the following as a primer. The second stage cDNA is It was obtained by the method described in the paper by Gfibler and Hoffmann (1983).

A kinase-treated EcoRI linker is ligated to the heavyweight double-stranded cDNA and Pst To connect the I linker to the light chain duplex cDN^, both of the possible To protect the site, first treat it with EcoRI or PstI methylase. ), then pUC9 ( Vieira et al. (1982)) to create E. coli TG2 strain (Gibso (1984)) was transformed.

Contains the gene encoding mouse HMFGI VH (I[oVHHllFGl) colony and mouse HMFGI Vk (MoVkHMFGl) = 2-do Colonies containing the gene for HMFGl VH and Vk first Colony hybridization using two types of probes consisting of step 32p-labeled cDNA It was identified using the cation method. Positive colonies were characterized by plasmid preparation and then Ec This was revealed by oRI or PstI digestion and 1.5% agarose gel analysis. the above The insert fragment (approximately 450 bp) containing the entire gene was transferred to a lid 13mp1g (Norra subclonin at the EcoRI site or PstI site of der et al. (1983)). I clicked. This operation yielded clones with different insertion directions; , by the dideoxy chain terminator method (Sanger et al. (1977)) Sequencing of the entire insert became easy.

Mature variable region gene MoVHHMFG1 and i[oVkHl[FGl] base sequence The sequences and the amino acid sequences translated from them are shown in FIGS. 1 and 2. 450bp Although not shown in the figure, the inserted fragment contains a signal sequence, an untranslated sequence, and a linker. was included.

2. Transfer the mouse HMFGI CDR onto the human framework region. The techniques required for uno are described by Jones et al. (1986) and Verhoeyen et al. 88), Japanese text of Riechmann et al. (1988) and European Patent Publication No. 239 No. 400 (linter).

a) light chain The basic construct used for human light chain modification was l[13mp9HuVkLYs (Riec hmann et al. (1988)), but this is a Bence-Jones protein. REI (a sequence based on the framework region of the light chain variable region of Epp et al. (1974)) Contains a framework area with columns.

Each CDR in this construct (Fig. 5a) is coded for each CDR of the chain in MFGI. (At both ends of each CDH are 12 base nuclei encoding the corresponding human framework residues. using oligonucleotides VI, VII and VIII (linked with leotide) The substitution was performed using site-directed mutagenesis. These oligonucleotides are shown in Figure 11. The above mutation was introduced according to the method of Riechmann et al. (1988). went. The obtained modified human light chain variable region gene (■uVkHMFG1) is shown in Figure 13. Shown below.

b) Heavyweight The modified human heavyweight variable region genes were obtained by "Donobo" synthesis. Jone above In the experiments described in other Japanese publications, the heavy chain CDRs of the oath animals were changed to human NEW heavy chain CDRs. The framework area has been populated above. Verhoeyen et al. (1988 ) and Riechmann et al. (1988) show that the human frame Work has shown that the animal-derived CDRs are similar in conformation to the original modont antibody. It is important to be able to maintain this conformation, and certain CDR-frames teamwork interaction is of critical importance. Therefore, the oath animal framework The greater the dissimilarity between the human framework sequence and the human framework sequence, the more likely it is that CDR import will occur. The chances of it happening are lower.

Heavy chain variable region amino acid sequence of mouse H1l [FGl (Figure 1) and human NEW (V (as described in Erhoeyen et al. (198g)). The difference between these individual framework areas is 44%. Subgroup I ( Kabat et al. (1987)), human variable regions belonging to subgroup A. A higher degree of homology is observed when compared with VHNEf.

Therefore, we found that the subgroup human heavies containing the HMFGI heavies CDRs We decided to synthesize variable region genes. We have variable human stalwart subgroups The consensus sequence (consensus 5 sequences) for the region is It was designed based on the sequence information of this subgroup described in T et al. (1987). . The optimal codon usage is the mouse constant region gene (this gene is derived from mouse myeloma cells). (expressed in the cell system).

HMF (dVH framework sequence and this human vH subgroup common sequence) The difference with HuVHIcon was only 14%. Obtained modified gene was named HuVHIconHFMGl and is shown in FIG. This gene combination The configuration will be explained separately in item (c) below. Newly synthesized gene HuVHrco Construct 1113mp9HuVHLYs (Verhoey (1988)) and vector M13mp9Hu VHIconHFMG1 was obtained (see Figure 4a).

3. Construction of modified human antibody genes in expression vectors The next step was er et al. (1983) with the use of the mouse heavy enhancer-IgEnh. It is. This enhancer is lkbXba of plasmid psV-V x1 Contained in the l fragment. To confer enhancer activity, this lkb 700bp of Xbal fragment Xbal/EcoRI subfragment is sufficient.

Another source of enhancers is the plasmid psVneoHuVkPLAP (see Figure 58). ). E. coli containing this plasmid variant is subject to the Budapest Treaty. It was deposited as NCTC 12390 on April 19, 1990. to this deposit Such a plasmid further contains the human chain constant region gene (BamHI site ).

The modified human genes prepared in items 2(a) and 2(b) above are transferred to the M13 vector. It was excised as a HindlII-BamHI fragment. Key variable region genetics A vector based on psV2gpt (Mulltngan et al. (1981)) The light chain variable region gene was cloned into psV2neo (Southern (1981)) into an expression vector. These are both immunoglobulins Contains the key enhancer - IgEnh. psV2gpt antibody expression vector (see Figures 4b to 40), Xbal/EcoRI enhancer-containing Cloning the fragment into the unique EcoRI site of the psV2gpt vector (Supplemented with the single-stranded part of the fragment's I linker).

In the psVneo antibody expression vector (see Figures 5a and 5b), lkb The Xbal enhancer-containing fragment was isolated from pUc12 (Vieira et al. (19 82)) to create plasmid pUc12-IgEnh (Fig. ) was obtained. This enhancer is then 700bp EcoRI/BindII can be excised as an I fragment (enhancers in either orientation are active) possible).

This 700bp EcoRI/HindIII fragment is a plasmid psV Present in neoHuVkPLAP. This plasmid psVneoHuVkP Using LAP, the HuVklll[FG1-containing fragment described in item 2(a)] (See Figures 5a and 5b). Existed in the original psVneo The 1ndI site has been removed. There is no need to actually select neo, so it is light. It is also possible to use psV2gpt as a vector for chain expression.

HuVHIconHl [FG1 gene was converted into human 71 constant region (Takahashi (1982)) and first pBGs], 8 (Spratt et al. (198 6) Cloned as an 8kb HindIII fragment into the BindIII site of and then cloned into psV2gpt as a BamHI fragment. I clicked. Here, the Japanese text of Takahashi et al. (1982) has an error in Figure 1. I would like to point out that. That is, the last two parts (3') are located next to BamHI. HindlII and not vice versa. This is true for F1 anagan et al. (1982).

The BuVkHMFG1 gene was cloned into the same (BamHI fragment was linked to the human C-group constant region (Hieter et al. (1980)) (Figures 5b and 8 reference). The human Ck source used in Figure 8 was described in Japanese by Hieter et al. (1980). It is. Derived from fetal DNA (cloned into γCh28 vector system) 12kb BamHI; fragment was inserted into the BamHI region of plasmid pBR322. We subcloned the human variable region genes of subgroup E (Kabat et al. 1987)) together with the VHHMGFI CDR (Fig. 1):::I-do gene It was decided to synthesize. Briefly, the synthetic gene is derived from the existing M13mp 9. The HuVHLYS gene in the HuVHLYS vector can be replaced. Designed by sea urchins.

A newly synthesized gene! [Cloned as pnI-3alI fragment Inject the KpnI site into the appropriate site of the lid 13++p9 uVHLYs so that and 5alI sites were introduced.

This gene sequence is named as described above in item 2(b) and is shown in FIG.

M1311p9HuVHLYS by this gene (Verhoeyen et al. (19 88), see Figure 48). 5' and 3' extensions were added to the gene. The 5' extension is 37b of the leader intron. p and leader exon (similar to that in M13mp9HuVHLYS). It contains the latter half of 1lbp and contains the KpnI site correctly at the 5' end. . The 3' extension consists of 38 bases of untranslated nucleotides (M13mp9HuVHLYS (similar to that in ) and ends with a 5alI site.

By site-directed mutagenesis using oligonucleotides I and II, l[13mp Modify 9HuVHLYs to introduce KpnI and 5alI sites at appropriate sites (See FIGS. 48 and 10). This vector is l[13mp9HuVHLYs It was named (1,S). This allows the KpnI-3alI fragment to be HuVHIconHFMG1 gene l113mp9HuVHLYs (K, S ) cloning into vectors is now possible.

For practical reasons, we synthesized the above gene as three fragments (cassettes). They then decided to assemble them into one complete gene.

Each fragment contains one of the three VHHFMGI CDRs, ( Easy access using existing or newly introduced unique restriction enzyme sites (see Figure 3a). can be cloned or removed. 5' and 3' ends of each fragment pE was extended to create 1ndIII and BamHI sites, respectively. BL9 (Dente et al. (1983)).

The coding strand of each fragment is divided into oligonucleotides with an average length of 33 bases. I divided it. The same can be said for the non-coding strand, if the oligonucleotide is This was done so that there was approximately 50% overlap with the oligonucleotide of the leading strand.

The sequences of each fragment and the oligonucleotides used in the construction are shown in Figure 3b, Figure 3. 0 and shown in Figure 3d.

Before constructing the fragments, add each synthetic oligonucleotide to facilitate ligation. It was necessary to phosphorylate the 5' end of Phosphorylation was performed as follows. Tomo Amounts of oligonucleotides (50 pmol) were pooled to form an 8 unit polynucleotide. 30-45 min at 37°C in 40 μl reaction buffer containing otidekinase. Kinase treated. The reaction was stopped by heating at 70°C for 5 minutes and ethanol precipitation. did.

7mM Tris-CI (pH 7,5), 10mM 2-mercaptoethanol Dissolve the above pellet in 30 μl of buffer containing SmM and perform annealing. ATP was added. The mixture was then placed in a 65°C water bath for 5 minutes and then After that, it was cooled to 30°C over 1 hour. The final concentration of MgCl2 was 10mM. I added it so that

Add TJ DNA ligase (2,5 units) and heat the mixture to 37°C for 30 minutes (or (overnight) at 16°C. After this time, the reaction mixture was heated at 70°C for 10 minutes. Eta Dissolve the pellet in the digestion buffer after nol precipitation! 1indIII and Bam Cut with 1l[I. Separate the mixture on a 2% agarose gel to ensure proper assembly. The fragments of the corresponding length of the cassette +7 were electrophoretically eluted and isolated. Ta.

Fragment (1, 2, 3) was added to pEMllN, 9 (HindIII/Bamt (cut with lI) to create vectors pUR4107゜p11R, respectively. 4108 and p! JR4109 was obtained. Sequence analysis of the insert (in both directions) I was surprised. Fragment 1 was digested with KpnI/XboI to create pUR4107. On the other hand, fragment 2 was digested with pUR4108 by XhoI/5acI digestion. They were then isolated from pU which had been cut with i[pnI/5acI. It was ligated with R4109 using the three-fragment ligation method. The obtained plasmid was called pUR4110. (see Figure 4a). Sequence analysis shows that the insert fragment is the desired HuVHIconH It was found that it contains the FMG1 gene. This gene is shown in Figures 4b and 4C. It was cloned into a psV2gpt-derived expression vector as shown in . vector p sVgptMoVHLYs-11oIgG1 (Verhoeyen et al. (198 8)) as the source of the psVgpt-based vector containing the IgEnh enhancer. Two expression plasmids were used: pSVgptHuVHIconHMFG1-HuI NSO of gGl and psVneoHuVkH revised Gl-Buck (Fig. 4c and b) Co-transfection into myeloma cells was performed after linearization treatment with PvuI. It was performed using the pneumoporation method (Potter et al. (1984)). Expressing the gpt gene product Transfectomas were incubated in mycophenolic acid-containing medium to select cells that Selected and screened for antibody production and anti-HMFG activity in ELISA assay. I did a ning.

Clones that were positive in both assays were obtained and purified by subcloning using the limiting dilution method. The best clones were assayed again for anti-HMFG activity and the highest producing clones were Lawns were grown in serum-free medium to produce antibodies.

6. Deposited plasmid The E. coli strain containing the plasmid used in the above procedure was as follows: As of July 11, 1990, National Co. It has been deposited in the collection of Type Cultures.

NCTC12411ni Plasmid I) SVgptHuV IconHMFG1-fluIgGl (deposited In the case of coli [12, TGI strain NCTC12412] E. coli mouth 2 containing plasmid psVneo-HuVkHMFG1-HuCk ．． TGI stock 7. Binding ability of modified human antibodies An effective method to demonstrate the binding ability of modified antibodies is to adsorb antigen onto a solid surface. We show that the modified antibodies have similar antibody dilution curves when conjugated to It is to do so. This curve was prepared using the parental mouse anti-HMFG antibody and the procedure described above. It was obtained using a modified human antibody as follows.

0.5 ml of 10% (w/V) i[280 tosyl-activated magnetic beads (Dyna One company, Wirral, UK, has developed an immunoassay for milk mucin (HliFGl). 106 units, where standard human serum shows 100-200 units/ml. ) was coupled. Breast mucin was obtained using the method of Burchell et al. (1987). Prepared from human milk. The above mucin concentration should be determined to have appropriate activity in the bead-based assay. It was chosen so that it can be obtained. The coupling reaction consists of 2.5 ml of 0.5 Phosphate buffered saline solution (pH 7) containing mucin in M borate buffer (pH 9,5) , 2, PBS) was added and the mixture was heated at 37°C for 22 hours with gentle shaking. Ta. Blocking of residual active sites was performed using PBSA (PBS 40.02% sodium azide). ) of 10% bovine serum albumin (abbreviated as BSA, manufactured by Sigma) and further incubated at 37° C. for 7 hours. samarium kobal After the beads were sedimented with a magnet, excess protein was washed away. Also for cleaning Buffer solution (0,111 potassium phosphate pH 8, 0, 0, 1% Tween 20. Wash 3 times in 0.5% BSA) and rinse buffer (PBS+O, ], % Washed 4 times in BSA, 091% merthiolate). Beads at 10% (W/ V) at a concentration (determined by dry weight analysis) in rinsing buffer.

The amount of antibody bound is determined by a 2-fold dilution series of the antibody sample (calibrated and prepared for the definitive one). It was measured from Add 50 μl samples in duplicate into microtiter wells and add 1% BSA/ 0.05% (W/V) Bea in PBSM (PBS + 0.01% merthiolate) The plate was incubated with 50 μl of the suspension for 1 hour at room temperature on a plate shaker. plus Using small samarium-cobalt magnets embedded in the Tick base material, Remove the liquid by settling the beads on the surface of the wells of the plate and add 150 μm PBSTM. Washed once with (PBSl [+0.15% Tween 20)]. After this, combine anti- 50111 diluted 1/1000 in a 1% BSA solution in PBSTM. Lucari phosphatase-conjugated goat anti-human IgG (H+L) (manufactured by Jackson) ) for 1 hour at room temperature. Beads were washed three times with PBSTII. Coloring The reaction was carried out using nitrophenyl phosphate in IM diethanolamine buffer (pH 9,8). The test was carried out using 200 μl of alkaline phosphatase substrate tablets manufactured by Sigma. . After transferring a predetermined amount of supernatant (usually 150 μl) to a flat bottom microtiter well, The optical density at 410 nm was measured using a plate reader manufactured by Tech. mouse anti The conjugated antibody used in the body assay was rabbit anti-mouse IgG (manufactured by Sigma). there were.

Antibody dilution curves for mouse and modified HMFGI antibodies are shown in FIG. 15. maximum join Activity was determined with large excess of antibody. Negative controls had no binding activity.

Antibody concentration in IIg/ml was determined by UV absorption measurement at 280 nm. Either Regarding the antibody, ljg/ml was also set as a dilution rate of 1. two curves are similar, indicating a significant and useful binding effect of the engineered antibodies of the invention.

References Arklie et al. (1981) -Int, J, Cancer, 28.23- p. 29 Burchell et al. (1987) - Cancer Res, 47. 5476 pages Dente et al. (1983) - Nucleic Ac1ds Re s, Il, pp. 1645-1655 Epp et al. (1974) - Eur, J, B iochem, 45. pp. 513-524 Flanagan et al. (1982) -Nature, pp. 300.709-713 Gendler et al. (1938) -J, Biol, Chew, 236. Pages 12820-12823 Gibson T (1984) - PhD thesis, University of Cambridge LIiB-M RcGubler et al. (1983) - Gene, 25. pp. 263-269 Ba1 e et al. (1988) - Lancet, 2. 1394 pages Hieter et al. (198 0) -Ce1l, 22. Pages 197-207 Jones et al. (1986) -N ature, 321. Pages 522-525 Kabat et al. (1987) -rse Quences of Proteins of Immunological InterestJ (published by the U.S. Department of Health and Human Services) page ix Mullingan et al. (1981) - Proc, Natn, Acad, Sci .

U, S, A,, 78. pp. 2072-2076 Neuberger et al. (1983) -ENBO Journal, 2. Pages 1373-1378 Norrander et al. (1983) - Gene, 26. Pages 101-106 Potter et al. (19 84) - PNAS, pp. 81.7161-7163 Riecbmann et al. (1 98g) - Nature, pp. 332.323-327 Sambrook et al. 1989) - rMolecular CloningJ 2nd edition (ColdSp ring Harbor Laboratory Press (-Knee York ) Sanger et al. (1977) - PNAS USA, 74. 5463 -Page 5467 Sau l et al. (1978) -J, Biol, Chew,... 253. pp. 585-597 5othern et al. (1981) -J, irises o1.　 Appl, Genetics, 1. Pages 327-345 5 Pratt et al. (1986) - Gene, 41. pp. 337-342 Taka hashi et al. (1982) - Ce11.29.671-679 Tay1or -Papadimitriou et al. (1981) -Int, J, Cancer.

28, pp. 17-21 Verhoeyen et al. (198g)-5science, 239.　1543- Page 1536 Vieira et al. (19g2) - Gene, pp. 19.259-268 Winter (1987) - European Patent Publication No. 239400 Xing et al. (199 0) - European Patent Publication No. 369816 code Chain length 5' ÷ - 11 Column - 11 ÷31 code Chain length 51 so---Sequence 1j---→31 code Chain length 51÷- m-sequence -11+3pBGs18-HuCk subclone in BamHI BamBI fragment gives pBGs18-Buck: mouse invariant γ 1 primer 5’ GAT AGA CAG ATG GGG GTG TCG TTT 3 '11: Primer unchanged in mouse 5’ AGA TGG ATA CAG T2CGTG CAG CAT 3’ I engineering: 5’ TGT for introducing KpnI site into HuVH leader intron CAT T UNICONI CA TAT 3' Engineering ■: To 5' of HuVHLYS gene 5' AAA TCT AT two units σGAA TAG for introducing the 5alI site of To 3’0ro0roo c ~ b?　0　Ma ー　−〜　I+l　F-1 Maximum binding amount (%) international search report ls+-nmw*l Awl, -1m No death? %. word. 14. .

FwlIIPCTMA/210 (-1 ~ -m +211 ・Pk1280Sl '11 International Search Report Continuation of front page (51) Int, C1,5 identification symbol Internal office reference number C07K 15/14 8517-4H15/28 8517-4H C12N 1/21 7236-4B //C07K 7106 Z 8318-4H71087537-4H GOIN 33153 D 8310-2J331574 Z 9015-2J (C12P 21108 C12R1:19) (C12N 1/21 C12R1:19) CO7K 99:00 I

Claims (30)

[Claims] 1. Amino acid sequence below: i) [There is an array] ii) [There is an array] iii) [There is an array] iv) [There is an array] v) [There is an array] vi) [There is an array] to human polymorphic epithelial mucin (PEM) conferred by one or more of Synthetic specific binding agents with specificity. 2. Amino acid sequence below: i) [There is an array] ii) [There is an array] iii) [There is an array] iv) [There is an array] v) [There is an array] vi) [There is an array] to human polymorphic epithelial mucin (PEM) conferred by one or more of A modified human antibody or modified human antibody fragment having the specificity of 3. The modified human antibody or modified human antibody fragment according to claim 2, which has the following CDRs: CDR1: [There is an array] CDR2: [There is an array] CDR3: [There is an array] Modified human antibodies or modified human antibodies containing one or more key variable regions incorporating body fragments. 4. The modified human antibody or modified human antibody fragment according to claim 2, which has the following CDRs: CDR1: [There is an array] CDR2: [There is an array] CDR3: [There is an array] Modified human antibodies or modified human antibodies containing one or more light chain variable regions incorporating body fragments. 5. The modified human antibody or modified human antibody fragment according to claim 2, one or more heavy chain variable regions and one or more light chain variable regions according to claim 4. A modified human antibody or modified human antibody fragment containing the above. 6. The modified human antibody or modified human antibody fragment according to claim 2 can be used as a modified human antibody or a modified human antibody fragment according to FIG. Modifications incorporating one or more heavy chain variable regions comprising the entire amino acid sequence shown in 2. Manufactured human antibodies or modified human antibody fragments. 7. The modified human antibody or modified human antibody fragment according to claim 2 can be used as a modified human antibody or a modified human antibody fragment according to FIG. Modifications incorporating one or more light chain variable regions comprising the entire amino acid sequence shown in 3. Manufactured human antibodies or modified human antibody fragments. 8. The synthetic specific binding agent according to any one of claims 1 to 7, a modified human antibody. body or modified human antibody fragment, the PEM is human milk fat globule (HMFG). Synthetic specific binding agents, modified human antibodies or modified human antibody fragments. 9.71,4 has the same specificity as the anti-HMFG monoclonal antibody HMFG1. Synthetic specific binding agents, modified human antibodies or modified human antibody fragments with isomerism. 10. The synthetic specific binding agent according to any one of claims 1 to 9, a modified human Stable host cell lines that produce antibodies or engineered human antibody fragments, and A foreign gene encoding a binding agent, a modified human antibody, or a modified human antibody fragment is introduced into the above cells. host cell line obtained by introducing it into the system. 11. 11. The stable host cell system according to claim 10, wherein the foreign gene comprises the following nucleic acid: Reotide sequence: i) [There is an array] ii) [There is an array] iii) [There is an array] iv) [There is an array] v) [There is an array] vi) [There is an array] A host cell line containing one or more of the following. 12. 11. The stable host cell system according to claim 10, wherein the foreign gene is A host cell line containing the entire nucleotide sequence shown in Figure 12. 13. 11. The stable host cell system according to claim 10, wherein the foreign gene is A host cell line containing the entire nucleotide sequence shown in FIG. 14. 11. The stable host cell system of claim 10, wherein the foreign gene is a) The following amino acid sequence: i) [There is an array] ii) [There is an array] iii) [There is an array] iv) [There is an array] v) [There is an array] vi) [There is an array] encode at least one of the following; and b) The above encoded amino acid sequence has specificity for PEM upon its expression. A protein framework that can function as a CDR with A host cell system that also encodes. 15. 11. The stable host cell system according to claim 10, wherein the foreign gene is A host cell line encoding the entire amino acid sequence shown in FIG. 16. 11. The stable host cell system according to claim 10, wherein the foreign gene is A host cell line encoding the entire amino acid sequence shown in FIG. 17. Plasmid pSVgpt-HuVHHMFGl-HuIgGl. 18. Plasmid pSVneo-HuVkHMFGl-HuCk. 19. Claims to production of synthetic specific binding agents, modified human antibodies or modified human antibody fragments Use of the plasmid according to claim 17 or claim 18. 20. Escherichia coli NCTC12411 strain. 21. Escherichia coli NCTC12412 strain. 22. Specificity against HmFG, such as that contained in E. coli NCTC12411 strain. A DNA sequence encoding a modified human antibody heavy chain variable region having isomerism. 23. Specificity against HMFG, such as that contained in E. coli NCTC12412 strain. A DNA sequence encoding a modified human antibody light chain variable region having isomerism. 24. Production using the expression vector contained in E. coli NCTC12411 strain. A modified human antibody heavy chain variable region having specificity for HMFG, which can be used for HMFG. 25. Production using the expression vector contained in E. coli NCTC12412 strain. A modified human antibody light chain variable region having specificity for HMFG, which can be used for HMFG. 26. An improvement comprising at least one of the variable parts according to claim 24 or claim 25. Manufactured human antibodies or modified human antibody fragments. 27. Synthetic specific binding according to any one of claims 1 to 9 or claim 26 Combination drugs, modified human antibodies, or modified human antibody fragments can be used to slow or slow the growth of cancer cells. are bound or incorporated into drugs that can be stopped or detected in the human body. Synthetic specific binding agents, modified human antibodies or modified human antibody fragments conjugated to agents that can be released . 28. Synthetic specific binding agent, modified human antibody or modified human antibody fragment according to claim 27 in a pharmaceutically acceptable carrier. 29. Manufacture of therapeutic drugs to save humans from cancer or in-vivo diagnostic tests for humans. Any one of claims 1 to 9 or claim 26 for producing a dispensable composition. Use of synthetic specific binding agents, modified human antibodies or modified human antibody fragments as described above. 30. A synthetic specificity according to claim 27 for a method of treating cancer or tomography in humans. use of synthetic binding agents, modified human antibodies or modified human antibody fragments.